Investigation On Melatonin Activating Ovarian Germline Stem Cells Function Via Macrophage Polarization Adjustment

Objective:To study the effect of Melatonin（MT）on the activation of endogenous Ovarian germinal stem cells（OGSCs）by regulating the polarization of Ovarian macrophages.Methods:A hypoxia model of RAW264.7 macrophages from mice in vitro is induced by cobalt chloride（CoCl₂）,then,the polarization state of macrophages under hypoxia environment is performed.Furthermore,the effect of MT on improvement of hypoxia and the regulation of polarization of macrophage Mφis explored via the hypoxia model.The effect of MT on the activation of mouse OGSCs is investigated by co-culture of ovarian and macrophages.1.Screening the appropriate concentration and reaction time during the process of CoCl₂inducing RAW264.7 macrophage hypoxia model.RAW264.7 macrophages are incubated with CoCl₂of multiple concentrations,and are grouped relying on the CoCl₂concentration,Control group,50μM CoCl₂group,100μM CoCl₂group and150μM CoCl₂group.The protein levels of Hypoxia Inducible Factor-1α（HIF-1α）and Vascular Endothelial Growth Factor（VEGF）are detected by Western blot.Enzyme Linked Immunosorbent Assay（ELISA）is used to detect Tumor Necrosis Factor alpha（TNF-α）,and nitric oxide（NO）detection kit is used to measure the NO content.To determine the optimal incubation time of CoCl₂in hypoxia modeling experiment.During the incubation process of RAW264.7 macrophages by CoCl₂,protein contents of HIF-1α,nuclear factor kappa-B（NF-κB）and phospho-NF-κB（p-NF-κB）are supervised by Western blot to determine the optimal incubation time of CoCl₂.2.Study on the role of MT in regulating the inflammatory cytokines release of RAW264.7 macrophage.MT concentrations of 0.01 m M,0.1 m M,1 m M,10 m M and100 m M are co-cultured with RAW264.7 macrophages after hypoxia induced by CoCl₂.The protein contents of HIF-1α,NF-κB and p-NF-κB in RAW264.7macrophages are also detected by Western blot.3.Study on the role of MT in the activation of ovarian reproductive stem cells by regulating RAW264.7 macrophage inflammatory factor release.The ovaries of Kunming mice are co-cultured with RAW264.7 macrophages for 21 days,and they are grouped as,blank group,hypoxic group（100μM CoCl₂）,and melatonin treatment group（100μM CoCl₂and 0.01 m M MT）.The levels of TNF-αand interleukin-10（IL-10）in cell culture medium are determined by ELISA.Western blot is used to detect the protein content of HIF-1α,NF-κB and p-NF-κB in RAW264.7macrophages,and the mouse vasa homolog（MVH）and octamer-binding protein-4（OCT-4）,and the difference in distribution of MVH and OCT-4 is observed by immunohistochemistry.Results:1.The optimal concentration and reaction time of CoCl₂-induced RAW264.7macrophage hypoxia model were screened.In a dose-dependent manner,50 to 150μM CoCl₂induced the increase of RAW264.7 HIF-1αand VEGF protein,TNF-αand NO content in macrophages（P<0.01）.The variation of above indexes was significant at 100μM CoCl₂.The results showed that the appropriate concentration of CoCl₂to induce hypoxia of RAW264.7 macrophages was 100μM,so 100μM CoCl₂was selected for subsequent experiments.After 100μM CoCl₂and RAW264.7macrophages were treated for 0,15,30 min and 1,2,4,8,12,24 h,Western blot analysis showed that HIF-1αexpression was the highest at about 8 h.NF-κB expression did not change over time.The expression level of p-NF-κB was obviously the highest at 30 min,so as to determine the subsequent MT dosage,the CoCl₂sample was collected after 8 h incubation when detecting HIF-1αchange,and the CoCl₂sample was collected after 30 min incubation when detecting p-NF-κB change.2.MT can reduce the release of RAW264.7 inflammatory factor induced by CoCl₂.MT co-incubated with hypoxic RAW264.7 macrophages significantly reduced the expression of HIF-1α:Western blot results showed that HIF-1αwas significantly decreased in 0.01 m M MT group compared with hypoxic model group（P<0.01）.Meanwhile,MT inhibited the inflammatory response of RAW264.7 macrophages:Western blot results showed that different concentrations of MT had no effect on NF-κB expression,but significantly inhibited the expression of p-NF-κB.Compared with hypoxia model group,the expression of p-NF-κB in 0.01m M MT group was significantly decreased（P<0.01）.3.When hypoxic macrophages were co-cultured with ovarian tissue,MT could improve the hypoxia of RAW264.7 macrophages induced by CoCl₂.The expression of HIF-1αwas significantly decreased in the treatment group compared with the hypoxia group（P<0.01）,and the expression of p-NF-κB was significantly decreased in the treatment group（P<0.01）,while the expression of NF-κB remained unchanged.The contents of TNF-αand IL-10 in macrophage ovarian co-culture were detected by ELISA.The results showed that TNF-αdecreased to a certain extent in the treatment group and hypoxia group,while IL-10 secretion increased to a certain extent in the treatment group and hypoxia group（P<0.05）.MT activates ovarian reproductive stem cells by inhibiting the RAW264.7macrophage releasing inflammatory factor.The results of Western blot showed that the expression of MVH and OCT-4 protein in the hypoxia group was significantly lower than that in the control group（P<0.01）.The protein expressions of MVH and OCT-4 in MT treatment group were higher than those in hypoxia group,and MVH treatment group was significantly higher than hypoxia group（P<0.01）.Immunohistochemical staining showed that the expression of MVH in hypoxia group was lower than that in control group（P<0.01）,and the expression of OCT-4 in hypoxia group was lower than that in control group（P<0.05）.The protein expressions of MVH and OCT-4 in MT treatment group were higher than those in hypoxia group（P<0.01）.Conclusions:CoCl₂can induce hypoxia in mouse RAW264.7 macrophages,MT can improve hypoxia and inflammation in RAW264.7 cells induced by CoCl₂,and its anti-inflammatory mechanism is inhibition of NF-κB phosphorylation.MT may activate the activity of ovarian reproductive stem cells by decreasing the secretion of TNF-αand promoting the secretion of IL-10 in RAW264.7 macrophages.Therefore,it is speculated that the mechanism by which MT activates the function of reproductive stem cells may be realized by regulating the polarization of resident ovarian macrophages.